COURSE OF MICROBIOLOGY 2014-2015 7. Biotechnology COURSE OF MICROBIOLOGY 2014-2015

I. What is Biotechnology? A. Biotechnology is the use of cells and/or biological molecules to solve problems or make useful products B. In a broad sense, many things are actually the products of biotechnology: 1. Bread, Beer/wine, cheese, yogurt C. Current examples include antibiotics, many pharmaceuticals, genetically modified organisms D. Modern biotechnology uses modern tools including computers, informatics, and recombinant DNA technology II. Recombinant DNA Recombinant DNA is the combining of DNA from two or more different sources into a single molecule A. Gel Electrophoresis is a method of separating DNA molecules based on their size 1. DNA has a negative charge from the phosphates so it will migrate toward the positive pole if an electric current is applied 2. Agarose is a polysaccharide that dissolves in water when heated and forms a solid matrix when cooled; it is basically highly refined agar

3. If we put DNA in an agarose gel and apply an electric current, 3. If we put DNA in an agarose gel and apply an electric current, DNA moves toward the positive pole at a rate inversely proportional to its size 4. DNA can be visualized with ethidium bromide B. DNA modifying enzymes 1. Restriction enzymes recognize specific DNA sequences and cut DNA within that sequence only 2. DNA Ligase joins the ends of linear DNA molecules 3. DNA Polymerases make new DNA molecules based on an existing template C. Making recombinant molecules combines the above enzymes and techniques 1. Recall that plasmids are small circular DNA molecules that replicate in bacteria and often contain antibiotic resistance genes 2. Plasmids can be used as vectors to carry foreign DNA sequences: 1. Plasmid and DNA of interest are digested with restriction enzymes 2. Both are run on an agarose gel to separate different fragments 3. Desired fragments (plasmid and insert) are excised from

gel and purified 4. Fragments are mixed together with DNA ligase, which joins the fragments 5. The resulting recombinant DNA molecule (plasmid+insert) are transformed into host bacteria (usually E. coli) 6. Bacteria containing the plasmid can be selected using antibiotic resistance 7. Once identified, colonies containing the recombinant plasmid can be grown to make large amounts of the plasmid (along with the insert DNA) 3. When we clone a gene, this is what we mean 4. Collections of cloned DNA fragments from a particular source are called libraries III. DNA profiling can be used to distinguish between individuals (including strains of microbes) A. DNA profiling examines the DNA of an individual (or clonal strain) so that it can be distinguished from other such entities B. This often takes one of two forms: 1. RFLP (Restriction Fragment Length Polymorphism)

2. PCR (Polymerase Chain Reaction) C. RFLP is the analysis of DNA fragments using restriction enzymes to cut the DNA 1. Restriction enzymes digested DNA is analyzed by Southern blot using a probe D. Specific DNA sequences can be amplified by PCR 1. PCR makes many copies of specific DNA sequences in a test tube 2. Involves DNA Polymerase, short DNA primers, and dNTPs 3. Here's how it works: a. Template DNA is heated to 95oC to denature (separate strands by breaking Hbonds) b. Temperature is reduced so that primers can bind to denatured DNA c. Temperature is raised to 72oC and DNA Polymerase extends the primers, copying the template sequence d. Return to step a (and repeat the whole process 30 times) 4. Each cycle of PCR doubles the number of molecules of the target sequence 5. After 30 cycles, there are more than 230 copies (that's a lot!) E. See examples used in class

IV. Genomics and Bioinformatics A. Genomics is the study of the entire DNA sequence of an organism B. Bioinformatics is the use of computer and information technology to analyze biological information (sequence data) C. The National Center for Biotechnology Information (NCBI) provides many useful tools and databases to store biological sequence information V. Genetic Engineering A. Genetic engineering refers to directed alterations of the genome of an organism B. Technically, selective breeding/domestication is genetic engineering in this sense C. Often used to refer to the insertion of one or more genes from one organism into another D. Modern genetic engineering uses recombinant DNA technology E. First commercial genetically engineered product was human insulin from Genentech (1982) 1. Insulin gene cloned into a plasmid and expressed in E. coli 2. Large cultures could then be grown and the protein extracted F. Now engineering of plants and animals is routine